The objectives of this project are to understand the nature of allosteric control and substrate specificity in the regulation of metabolic processes, to evaluate the role of subunit-subunit interactions, and to determine the effect of protein structure in terms of regulation and specificity of an enzyme. Studies will be conducted on two phosphoenolpyruvate carboxylases and two thiokinases found in the bacterium, Pseudomonas MA (ATCC No. 23319). The two phosphoenolpyruvate carboxylases are observed under different growth conditions, one characterized by being allosterically activated by NADH, is observed in methylamine grown cells, whereas an acetyl CoA activated enzyme is found under all other growth conditions tested. The two thiokinases can be described as a specific succinate thiokinase found under all growth conditions, and a non-specific malate thiokinase found only in methylamine grown cells. These enzymes will be purified to homogeneity and characterized in terms of their kinetic properties including substrate specificity, allosteric properties, and reaction mechanism. The subunit structures of the enzymes will be determined and compared in terms of subunit size and composition. Sequence homology between the two phosphoenolpyruvate carboxylases and between the two thiokinases will be determined by comparison of peptide maps. Attempts will be made to form hybrids between the two thiokinases and the two carboxylases in order to probe the effects of subunit interaction on the allosteric and catalytic properties of the enzymes. In addition, genetic experiments will be conducted to determine if the carboxylases or the two thiokinases are derived from a common gene product.